Method of treating human skin and a skin care composition for use in such a method

ABSTRACT

One aspect of the invention concerns a cosmetic method of treating human skin by delivering an estrogenic component to said skin. The method comprises applying to the skin a composition containing: (i) at least 5 μg/g of an estrogenic component selected from the group consisting of substances represented by the following formula (I) in which formula R 1 , R 2 , R 3 , R 4  independently are a hydrogen atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R 5 , R 6 , R 7  is a hydroxyl group; no more than 3 of R 1 , R 2 , R 3 , R 4  are hydrogen atoms; precursors capable of liberating a substance according to the aforementioned formula when used in the present method; and mixtures of one or more of the aforementioned substances and/or precursors; and (ii) a cosmetically acceptable vehicle. Other aspects of the invention relate to therapeutic methods of treating or preventing vaginal dryness or acne and a therapeutic method of promoting wound healing. Yet another aspect of the invention relates to a skin care composition comprising the aforementioned estrogenic component and a cosmetically acceptable vehicle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/517,509, filed Jun. 13, 2005, which is the U.S. National Phase of PCTApplication No. PCT/NL03/00420, filed Jun. 11, 2003, claiming priorityto EP 02077273.7 filed Jun. 11, 2002, which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a cosmetic method of treating humanskin by delivering an estrogenic component to said skin. Moreparticularly, the invention is concerned with such a method thatcomprises applying to the skin a composition containing an estrogeniccomponent and a cosmetically acceptable vehicle.

Another aspect of the invention concerns a method of therapeutic methodof treating or preventing vaginal dryness or acne and a therapeuticmethod of promoting wound healing, which methods comprise the deliveryof an estrogenic component to vaginal or skin epithelium

Yet another aspect of the invention relates to a skin care compositioncomprising an estrogenic component and a cosmetically acceptablevehicle.

BACKGROUND OF THE INVENTION

The human skin consists of two major layers, the bottom thicker layer(dermis) and the top thinner layer (epidermis). Dermis is the layerwhich provides the strength, elasticity and the thickness to the skin.

The main cell type of the dermis is the fibroblast, which is responsiblefor synthesis and secretion of all the dermal matrix components such ascollagen, elastin and glycosaminoglycans. Collagen provides thestrength, elastin the elasticity and glycosaminoglycans the moistnessand plumpness of the skin. With ageing, the thickness of the dermallayer is reduced and this is believed to be partially responsible forthe formation of wrinkles in ageing skin. The top layer of human skin orthe epidermis which provides the resilience and the barrier propertiesof the skin, is composed of many different cell types includingkeratinocytes, melanocytes and langerhans cells. Keratinocyte is themajor cell type of the epidermis (75-80% of the total number of cells inthe human epidermis). Richards et al. reported that estrogen stimulatessecretion of a protein, prolactin, by human dermal fibroblast cells andthat prolactin then stimulates proliferation of keratinocytes (Richardset al., Human Dermal Fibroblasts Express Prolactin In Vitro., J. Invest.Dermatol. (1996), 106: 1250).

Estrogens and synthetic compounds which act like estrogens are known toincrease the thickness of the dermal layer and to reduce wrinkleformation in the ageing skin. The changes in the skin such as skindryness, loss of skin elasticity and plumpness occurring after menopauseis attributed to the lack of estrogen production. Estrogen therapyprevents or slows down many of these changes associated with ageing skin(Creidi et al., “Effect of a conjugated estrogen cream on ageing facialskin”, Maturitas, (1994) 19, p. 211). Some of the effects of estrogen onskin include: increase in skin thickness and disappearance of finewrinkles, increase of the mitotic rate of the epidermis, reduction inthe size and activity of the sebaceous gland, slow down of the rate ofhair growth, stimulation of collagen turnover and increase in theproduction of hyaluronic acid and glycosaminoglycan synthesis of thefibroblasts (Pugliese, Menopausal skin, Skin Inc., March/April 1994: p69-77).

Schmidt et al. report on the effects on ageing skin of the face ofperimenopausal females treated with a 0.3% estriol cream or with a 0.01%estradiol cream for 6 months (Schmidt et al., “Treatment of skin ageingsymptoms in perimenopausal females with estrogen compounds. A pilotstudy”, Maturitas (1994), 29(1), 25-30). Both treatment groups werefound to show improvement of the various skin ageing symptoms at the endof treatment. The effects of the group treated with topical estriol weredeemed to be slightly superior with regard to their extent and onset.

Shah et al. (“Estrogen and skin. An overview”, Am J Clin Dermatol(2001); 2(3):143-150) report that topical and systemic estrogen therapycan increase the skin collagen content and therefore maintain skinthickness. In addition, it is said that estrogen maintains skin moistureby increasing acid mucopolysaccharides and hyaluronic acid in the skinand possibly maintaining stratum corneum bather function. Furthermore itis observed that estrogen may increase cutaneous wound healing byregulating cytokine levels as topical estrogen has been found toaccelerate and improve wound healing in elderly men and women.

In a review article by Sitruk-Ware et al. (“Topical hormonal treatmentand urogenital atrophy”, Schweiz Rundsch Med Prax (1997) August 13;86(33):1245-1248) it is stated that local estrogen therapies arerecommended for the treatment of complaints due to vulvar and vaginalatrophy. Estrogens specifically mentioned in the review article includeestrone, promestriene, estradiol and estriol.

SUMMARY OF THE INVENTION

The present invention provides a particularly effective method ofimproving or preventing the condition of wrinkled, lined, dry, flaky,aged or photodamaged skin and of improving skin thickness, elasticity,flexibility and plumpness, which method includes applying to the skin acomposition that contains an estrogenic component and a cosmeticallyacceptable carrier. The present invention encompasses a cosmetic methodof increasing fibroblast and epidermal skin cell proliferation in humanskin by applying to the skin the inventive composition. The presentmethod may be applied to human skin which is already dry, flaky, lined,wrinkled, aged, photodamaged, or to healthy skin to prevent or reducesuch deteriorative changes. The invention also concerns a therapeuticmethod of treating or preventing vaginal dryness as well as a method ofpromoting wound healing, which methods comprise the delivery of thecomposition as described herein before to the vaginal or skinepithelium.

The inventors have unexpectedly found that topical application of aspecial group of estrogenic substances produces surprisingly goodresults in terms of elasticity and firmness of the skin, wrinkle depthand pore sizes and/or skin moisture. These special estrogenic substancesare represented by the following formula

in which formula R₁, R₂, R₃, R₄ independently are a hydrogen atom, ahydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R₅, R₆,R₇ is a hydroxyl group; no more than 3 of R₁, R₂, R₃, R₄ are hydrogenatoms.

A known representative of this group of estrogenic substances is 1,3,5(10)-estratrien-3,15α,16α,17β-tetrol, also known by the names ofestetrol, oestetrol and 15α-hydroxyestriol. Estetrol is an estrogen thatis produced by the fetal liver during human pregnancy. Unconjugatedestetrol levels in maternal plasma peak at about 1.2 ng/ml at termpregnancy and are about 12 times higher in fetal than in maternal plasma(Tulchinsky et al., 1975. J. Clin. Endocrinol. Metab., 40, 560-567).

In 1970, Fishman et al., “Fate of 15α-hydroxyestriol-³H in Adult Man”, JClin Endocrinol Metab (1970) 31, 436-438, reported the results of astudy wherein tritium labeled 15α-hydroxyestriol (estetrol) wasadministered intravenously to two adult women. It was found that theestetrol was rapidly and completely excreted in urine as theglucosiduronate and that virtually no metabolism except for conjugationtook place.

Between 1975 and 1985 several researchers have investigated theproperties of estetrol and reported on its estrogenic potency anduterotrophic activity. The most relevant publications that were issuedduring this period are mentioned below:

-   -   Levine et al., 1984. Uterine vascular effects of estetrol in        nonpregnant ewes. Am. J. Obstet. Gynecol., 148:73, 735-738:        “When intravenously administered in nonpregnant ewes, estetrol        is 15 to 30 times less potent than estriol and 17β-estradiol in        uterine vasodilation”.    -   Jozan et al., 1981. Different effects of oestradiol, oestriol,        oestetrol and of oestrone on human breast cancer cells (MCF-7)        in long term tissue culture. Acta Endocrinologica, 98, 73-80:        “Estetrol agonistic potency is 2% of the magnitude observed for        17β-estradiol in in vitro cell proliferation”.    -   Holinka et al., 1980. Comparison of effects of estetrol and        tamoxifen with those of estriol and estradiol on the immature        rat uterus. Biol. Reprod. 22, 913-926: “Subcutaneously        administered estetrol has very weak uterotrophic activity and is        considerable less potent than 17β-estradiol and estriol”.    -   Holinka et al., 1979. In vivo effects of estetrol on the        immature rat uterus. Biol. Reprod. 20, 242-246: “Subcutaneously        administered estetrol has very weak uterotrophic activity and is        considerable less potent than 17β-estradiol and estriol”.    -   Tseng et al., 1978. Heterogeneity of saturable estradiol binding        sites in nuclei of human endometrium. Estetrol studies. J.        Steroid Biochem. 9, 1145-1148: “Relative binding of estetrol to        estrogen receptors in the human endometrium is 1.5% of        17β-estradiol”.    -   Martucci et al., 1977. Direction of estradiol metabolism as a        control of its hormonal action-uterotrophic activity of        estradiol metabolites. Endocrin. 101, 1709-1715: “Continuous        administration of estetrol from a subcutaneous depot shows very        weak uterotrophic activity and is considerably less potent than        17β-estradiol and estriol”.    -   Tseng et al., 1976. Competition of estetrol and ethynylestradiol        with estradiol for nuclear binding in human endometrium. J.        Steroid Biochem. 7, 817-822: “The relative binding constant of        estetrol binding to the estrogen receptor in the human        endometrium is 6.25% compared to 17β-estradiol (100%)”.    -   Martucci et al., 1976. Uterine estrogen receptor binding of        catecholestrogens and of estetrol        (1,3,5(10)-estratriene-3,15alpha,16alpha, 17beta-tetrol).        Steroids, 27, 325-333: “Relative binding affinity of estetrol to        rat uterine cytosol estrogen receptor is 0.5% of 17β-estradiol        (100%). Furthermore, the relative binding affinity of estetrol        to rat uterine nuclear estrogen receptor is 0.3% of        17β-estradiol (100%)”.

All of the above publications have in common that the authors haveinvestigated the estrogenic potency of estetrol. Without exception theyall conclude that estetrol is a weak estrogen. In some of the citedarticles the estrogenic potency of estetrol has been found to be muchlower than that of a relatively weak estrogen that is commonly used inpharmaceutical formulations, namely 17β-estradiol With these findings inmind, it is not surprising that the interest in estetrol has dwindledsince the early eighties and that no publications on the properties ofestetrol have been issued since.

In view of the low estrogenic potency of the estetrol-like substancesthat are employed in the present method, it is surprising that thesesubstances may effectively be used in the treatment of human skin.Although the inventors do not wish to be bound by theory, it is believedthat this efficacy may be related to the ability of the these substancesto penetrate through the skin and the relatively high in vivo half-lifeof these substances in comparison to e.g. 17β-estradiol and estriol.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention concerns a cosmetic method of treating humanskin by delivering an estrogenic component to said skin, the methodcomprising applying to the skin a composition containing:

(i) at least 5 μg/g of an estrogenic component selected from the groupconsisting of: substances represented by the following formula

in which formula R₁, R₂, R₃, R₄ independently are a hydrogen atom, ahydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R₅, R₆,R₇ is a hydroxyl group; no more than 3 of R₁, R₂, R₃, R₄ are hydrogenatoms;precursors capable of liberating a substance according to theaforementioned formula when used in the present method; andmixtures of one or more of the aforementioned substances and/orprecursors; and(ii) a cosmetically acceptable vehicle.

The term “estrogenic component” as used throughout this documentencompasses substances that are capable of triggering an estrogenicresponse in vivo, as well as precursors that are capable of liberatingsuch an estrogenic component in vivo when used in accordance with thepresent invention. In order for estrogenic components to trigger such aresponse they normally have to bind to an estrogen receptor, whichreceptors are found in various tissues within the mammalian body. It isnoted that the present invention not only encompasses the use ofestrogenic substances specifically mentioned in this application, butalso metabolites of these hormones that display comparable in vivofunctionality. In this context it is observed that, for instance,estriol is a metabolite of 17β-estradiol.

The terminology “delivering the estrogenic component to the skin”relates to the application of said estrogenic component to the surfaceof the skin, in particular topical application or transdermalapplication. Topical application suitably includes the application ofe.g. salves, lotions and creams to the skin surface. Transdermalapplication encompasses the fixation to the skin epithelium oftransdermal patches that contain the present composition.

An important benefit of the present method resides in the ability of thepresent estrogenic components to enhance proliferation of fibroblastsand/or epidermal cells. Consequently, in a preferred embodiment, thepresent composition is applied in an effective amount to enhanceproliferation of fibroblasts and/or epidermal skin cells. Enhancedproliferation will induce a “rejuvenation” of the skin as demonstratedby increased elasticity and firmness of the skin, reduced wrinkle depthand pore sizes and/or increased skin moisture

On a macroscopic level the benefits of the present method manifestthemselves in the form of improved skin thickness and/or skinelasticity, provided the composition is applied in an effective amount.A particularly preferred embodiment of the present invention relates toa cosmetic method, wherein the present composition is applied in aneffective amount to improve or prevent the condition of wrinkled, lined,dry, flaky, aged or photodamaged skin.

Although the present invention is perfectly suitable for treatinghypoestrogenic subjects, e.g. (peri-)menopausal and post-menopausalfemales, it is preferred not to apply the present estrogenic componentto the skin in sufficient quantities to significantly suppress symptomsof hypoestrogenism, such as osteoporosis and climacteric symptoms (e.g.hot flushes, palpitations and mood disturbances). The latter symptoms ofhypoestrogenism are more effectively treated by e.g. oral orsubcutaneous administration.

The present estrogenic substances are distinct from both the estrogensthat are commonly applied in pharmaceutical formulations (e.g.17α-ethinyl estadiol and 17β-estradiol) in that they contain at least 4hydroxyl groups. The present substances are particularly special in thatthe 5 membered ring in the steroid skeleton comprises 3 hydroxylsubstituents rather than 0-2.

Known estrogens that contain at least 4-hydroxyl groups and derivativesthereof are:

1,3,5(10)-estratrien-2,3,15α,16α,17β-pentol 2-methyl ether

1,3,5(10)-estratrien-2,3,15α,16α,17β-pentol 2-methyl ether

1,3,5(10)-estratrien-2,3,16α,17β-tetrol

1,3,5(10)-estratrien-3,4,16α,17β-tetrol 4-methyl ether

1,3,5(10)-estratrien-3,15α,16α,17β-tetrol

1,3,5(10)-estratrien-3,15α,16α,17β-tetrol tetra acetate

1,3,5(10)-estratrien-3,15β,16β,17β-tetrol tetra acetate

Preferably, the estrogenic substance applied as the active component inthe present composition is a so called biogenic estrogen, i.e. anestrogen that occurs naturally in the human body, or a precursorthereof. Because biogenic estrogens are naturally present in the fetaland female body, side-effects are not expected to occur, particularlynot if the serum levels resulting from the exogenous administration ofsuch estrogens do not exceed naturally occurring concentrations. Sinceestetrol serum levels in the fetus are several times higher than thosefound in pregnant females and knowing that the fetus is particularlyvulnerable, estetrol is deemed to be a particularly safe biogenicestrogen.

In a preferred embodiment of the present invention the estrogenicsubstance contains 4 hydroxyl groups. Also, in the aforementionedformula, R₁ preferably represents a hydrogen atom. In said formulapreferably at least 2, more preferably at least 3 of the groups R₁, R₂,R₃ and R₄ represent a hydrogen atom.

The estrogenic substances according to the formula encompass variousenantiomers since the carbon atoms that carry hydroxyl-substituents R₅,R₆ and R₇ are chirally active. In one preferred embodiment, the presentestrogenic substance is 15α-hydroxy substituted. In another preferredembodiment the substance is 16α-hydroxy substituted. In yet anotherpreferred embodiment, the substances is 17β-hydroxy substituted. Mostpreferably the estrogenic substances are 15α,16α,17β-trihydroxysubstituted.

In another preferred embodiment of the present invention R₃ represents ahydroxyl group or an alkoxy group. In another preferred embodiment thegroups R₁, R₂ and R₄ represent hydrogen atoms, in which case, if R₃, R₅,R₆ and R₇ are hydroxyl groups, the substance is 1,3,5(10)-estratrien-3,15,16,17-tetrol. A preferred isomer of the lattersubstance is 1,3,5 (10)-estratrien-3,15α,16α,17β-tetrol (estetrol).

The invention also encompasses the use of precursors of the estrogenicsubstances that constitute the active component in the present method.These precursors are capable of liberating the aforementioned estrogenicsubstances when used in the present method, e.g. as a result ofmetabolic conversion. These precursors are preferably selected from thegroup of androgenic precursors as well as derivatives of the presentestrogenic substances. Suitable examples of androgenic precursorsinclude androgens that can be converted into the present estrogenicsubstances through in vivo aromatisation. Examples of derivatives of thepresent estrogenic substances that can suitably be used as precursorsinclude such substances wherein the hydrogen atom of at least one of thehydroxyl groups has been substituted by an acyl radical of a hydrocarboncarboxylic, sulfonic acid or sulfamic acid of 1-25 carbon atoms;tetrahydrofuranyl; tetrahydropyranal; or a straight or branched chainglycosydic residue containing 1-20 glycosidic units per residue.

Typical examples of precursors which can suitably be used in accordancewith the invention are esters that can be obtained by reacting thehydroxyl groups of the estrogenic substances with substances thatcontain one or more carboxy (M⁺⁻OOC—) groups, wherein M⁺ represents ahydrogen or (akali)metal cation. Hence, in a particularly preferredembodiment, the precursors are derivatives of the estrogenic substances,wherein the hydrogen atom of at least one of the hydroxyl groups in saidformula has been substituted by —CO—R, wherein R is a hydrocarbonradical comprising from 1-25 carbon atoms. Preferably R is hydrogen, oran alkyl, alkenyl or aryl radical comprising from 1-20 carbon atoms.

The composition that is applied to the skin in accordance with thepresent invention suitably contains at least 10 μg/g of the estrogeniccomponent. Particularly satisfactory results can be obtained if thecomposition contains at least 30 μg/g, more preferably at least 50 μg/gand most preferably at least 100 μg/g of the estrogenic component. Forpractical reasons the present composition will usually not contain morethan 50 mg/g of the estrogenic component. Preferably said compositioncontains not more than 20 mg/g, more preferably not more than 10 mg/g ofthe estrogenic component.

The advantages of the present invention may be realised in skin as it isfound in different places of the human body. Particularly advantageousresults can be obtained by applying the composition to e.g. face, neck,shoulders, breast, buttocks (cellulitis) etc. In a preferred embodimentof the present method, the skin care composition is applied to facialskin or the skin of the neck.

In order to obtain quick and lasting results it is advisable to applythe present composition at least once a day during a period of at least3 days, particularly in case of topical application. Because transdermalapplication enables the relatively slow release of the presentestrogenic component over a longer period of time, transdermalapplication is preferably carried out with a frequency of at least oncea week. Topical application of the estrogenic component is the preferredmode of administration as it is uncomplicated, effective and producesessentially no undesirable side-effects.

The topical application of the present composition is preferably carriedout in such a way that virtually all of the composition that has beenapplied to the skin, is allowed to penetrate said skin. When appliedtopically, a small quantity of the present composition, for example from0.1 to 100 g, is applied directly to the skin, optionally from asuitable container or applicator and, if necessary, it is then spreadover and/or rubbed into the skin using the hand or fingers or a suitabledevice.

The benefits of the present invention are most pronounced when thecomposition is applied in the course of a longer term treatment.Therefore, the present method, preferably, comprises administering theestrogenic component for a period of at least 1 week, more preferably ofat least 3 weeks. The present method usually employs uninterruptedadministration of the estrogenic component during at least 10,preferably at least 20 days. The term “uninterrupted” as used in here,means that the estrogenic component is administered at relativelyregular intervals, with no (therapeutically) significant interruptions.Naturally, minor interruptions may occur that do not affect the overalleffectiveness of the present method, and indeed such aberrations areencompassed by the present invention. In a preferred embodiment, andmore arithmetically, the administration regimen is deemed to beuninterrupted if the longest interval between 2 subsequentadministrations is not more than 3.5 times as long as the averageinterval. Even more preferably said longest interval is not more than2.5 times, most preferably not more than 1.5 times as long as theaverage interval.

Another aspect of the invention relates to a method of treating orpreventing vaginal dryness, wherein the method comprises applying to thevaginal epithelium a composition containing:

(i) at least 5 μg/g of the present estrogenic component; and

(ii) a cosmetically acceptable vehicle.

A further aspect of the invention relates to a method of promoting woundhealing, wherein the method comprises applying to the wounded tissue orto tissue in the vicinity of the wound a composition containing:

(i) at least 5 μg/g of said estrogenic component; and

(ii) a cosmetically acceptable vehicle.

Yet another aspect of the invention relates a method of treating orpreventing acne, wherein the method comprises applying to the skin thatis affected by acne or that is at risk of being affected by acne acomposition containing:

(i) at least 5 μg/g of said estrogenic component; and

(ii) a cosmetically acceptable vehicle.

The preferred embodiments discussed herein before in relation to thetreatment of skin equally apply to the present method of treating orpreventing vaginal dryness and the method of promoting wound healing. Inthe latter methods continuous administration of the present compositionduring prolonged periods of time may not be necessary, particularly notin case of wound healing. Indeed, the present method of promoting woundhealing and, to a lesser extent, the method of treating vaginal drynessmay suitably employ administration on demand.

Yet another aspect of the invention relates to a skin care compositionfor topical administration, in accordance with the method describedherein before, said composition containing:

(i) at least 5 μg/g of the estrogenic component; and

(ii) a cosmetically acceptable vehicle.

The composition according to the invention comprises a cosmeticallyacceptable vehicle to act as a diluant, dispersant or carrier for theestrogenic component, so as to facilitate its distribution when thecomposition is applied to the skin. Vehicles other than or in additionto water can include liquid or solid emollients, solvents, humectants,thickeners and powders. Particularly suitable nonaqueous carriersinclude polydimethyl siloxane and/or polydimethyl phenyl siloxane.Especially desirable are mixtures of low and high viscosity silicones.These silicones are available from the General Electric Company undertrademarks Vicasil, SE and SF and from the Dow Corning Company under the200 and 550 Series. The amounts of silicone which can be utilised in thecompositions of this invention can range anywhere from 5% to 95%,preferably from 25% to 90% by weight of the composition. Thecosmetically acceptable vehicle will usually form from 5% to 99.9%,preferably from 25% to 95% by weight of the composition, and can, in theabsence of other cosmetic adjuncts, form the balance of the composition.Preferably, the vehicle is at least 80 wt. % water. Preferably, waterconstitutes at least 50 wt. % of the present composition, mostpreferably from 60 to 80 wt. %, by weight of the composition.

The skin care composition of the present invention may contain an oil orlipid material, together with an emulsifier, to provide either awater-in-oil emulsion or an oil-in-water emulsion, depending largely onthe average hydrophilic-lipophilic balance (HLB) of the emulsifieremployed. The present compositions include sunscreens. Sunscreensinclude those materials commonly employed to block ultraviolet light.Illustrative compounds are the derivatives of PABA, cinnamate andsalicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone) can be used. Octylmethoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commerciallyavailable under the trademarks, Parsol MCX and Benzophenone-3,respectively. The exact amount of sunscreen employed in the emulsionscan vary depending upon the degree of protection desired from the sun'sUV radiation.

Emollients can advantageously be incorporated into the compositions ofthe present invention. Levels of such emollients may range from 0.5% to50%, preferably from 5% and 30% by weight of the total composition.Emollients may be classified under such general chemical categories asesters, fatty acids and alcohols, polyols and hydrocarbons. Esters maybe mono- or di-esters. Acceptable examples of fatty di-esters includedibutyl adipate, diethyl sebacate, diisopropyl dimerate, and dioctylsuccinate. Acceptable branched chain fatty esters include 2-ethyl-hexylmyristate, isopropyl stearate and isostearyl palmitate. Acceptabletribasic acid esters include triisopropyl trilinoleate and trilaurylcitrate. Acceptable straight chain fatty esters include laurylpalmitate, myristyl lactate, and stearyl oleate. Preferred estersinclude coco-caprylate/caprate (a blend of coco-caprylate andcoco-caprate), propylene glycol myristyl ether acetate, diisopropyladipate and cetyl octanoate. Suitable fatty alcohols and acids includethose compounds having from 10 to 20 carbon atoms. Especially preferredare such compounds such as cetyl, myristyl, palmitic and stearylalcohols and acids.

Among the polyols which may serve as emollients are linear and branchedchain alkyl polyhydroxyl compounds. For example, propylene glycol,sorbitol and glycerin are preferred. Also useful may be polymericpolyols such as poly-propylene glycol and polyethylene glycol. Butyleneand propylene glycol are also especially preferred as penetrationenhancers. Exemplary hydrocarbons which may serve as emollients arethose having hydrocarbon chains anywhere from 12 to 30 carton atoms.Specific examples include mineral oil, petroleum jelly, squalene andisoparaffins.

Another category of functional ingredients that may be employed in thecosmetic compositions of the present invention are thickeners. Athickener will usually be present in amounts anywhere from 0.1 to 20% byweight, preferably from about 0.5% to 10% by weight of the composition.Exemplary thickeners are cross-linked polyacrylate materials availableunder the trademark Carbopol from the B.F. Goodrich Company. Gums may beemployed such as xanthan, carrageenan, gelatin, karaya, pectin andlocust beans gum. Under certain circumstances the thickening functionmay be accomplished by a material also serving as a silicone oremollient. For instance, silicone gums in excess of 10 centistokes andesters such as glycerol stearate have dual functionality.

Powders may also be incorporated into the composition of the invention.These powders include chalk, talc, kaolin, starch, smectite clays,chemically modified magnesium aluminium silicate, organically modifiedmontmorillonite clay, hydrated aluminium silicate, fumed silica,aluminium starch octenyl succinate and mixtures thereof.

Other adjunct minor components may also be incorporated into the presentcompositions. These ingredients may include colouring agents, opacifiersand perfumes. Amounts of these other adjunct minor components may rangeanywhere from 0.001% up to 20% by weight of the composition.

The topical skin care composition of the invention can be formulatede.g. as a lotion, a cream or a salve (e.g. a gel). The composition canbe packaged in a suitable container to suit its viscosity and intendeduse by the consumer. For example, a lotion or a cream can be packaged ina bottle or a roll-ball applicator, or a propellant-driven aerosoldevice or a container fitted with a pump suitable for finger operation.When the composition is a cream or a salve, it can simply be stored in anon-deformable bottle or squeeze container, such as a tube or a liddedjar. The composition may also be included in capsules such as thosedescribed in U.S. Pat. No. 5,063,507, incorporated by reference herein.The invention accordingly also provides a closed container containing acosmetically acceptable composition as herein defined.

The present invention is further illustrated by the following examples,which, however, are not to be construed as limiting. The featuresdisclosed in the foregoing description, in the following examples and inthe claims may, both separately and in any combination thereof, bematerial for realising the invention in diverse forms thereof.

EXAMPLES Example 1

Established competitive steroid binding assays were used to determinethe relative binding affinity of estetrol (E4), as compared to17α-ethinylestradiol(EE) and 17β-estradiol (E2), to human EstrogenReceptor (ER) α- and β-forms.

The method employed was adapted from the scientific literature anddescribed in detail by Osboum et al. (1993, Biochemistry, 32,6229-6236). Recombinant human ERα and ERβ proteins were purified fromtransfected Sf9-cells. The in vitro assays involved the use of eitherERα or ERβ proteins and [³H]E2, at a fixed concentration of 0.5 nM, asthe labeled ligand. Recombinant human ERα or ERβ proteins were dissolvedin binding buffer (10 mM Tris-HCL, pH 7.5, 10% glycerol, 1 mM DTT, 1mg/ml BSA) and duplicate aliquots were then incubated with [³H]E2 at afinal concentration of 0.5 nM, together with a vehicle control (0.4%DMSO), or the same amount of vehicle containing increasingconcentrations of unlabeled steroid ligands as competitors. Afterincubation for 2 h at 25° C., the unbound ligands were removed and theamounts of [³H]E2 bound to either ERα or ERβ proteins were measured. Theaverage amounts of [³H]E2 bound to either ERα or ERβ proteins at eachconcentration of competitor were used to make inhibition curves. IC50values were subsequently determined by a non-linear, least squaresregression analysis. Inhibition constants (Ki) were calculated using theequation of Cheng and Prusoff (Cheng et al., 1973, Biochem. Pharmacol.,22, 3099-3108), using the measured 1050 of the tested compounds, theconcentration of radioligand employed in the assay, and the historicalvalues for the Kd of the radioligand, which were established as 0.2 nMand 0.13 nM for ERα and ERβ, respectively.

Biochemical assay results for E4 are presented as the percent inhibitionof specific binding in three separate experiments (Table 1). Forcomparison of binding affinities of E4, EE and E2 to human ERα and ERβproteins, experimentally observed Ki values are shown in Table 2. Ascompared to both EE and E2, E4 shows substantially less bind affinityfor ERα and ERβ receptor forms (Table 2).

TABLE 1 Percent inhibition of specific binding to ERα and ERβ proteinsusing E4 as unlabeled steroid ligand and 0.5 nM [3H] as labeledcompetitor. Results of three separate experiments are shown. Percentinhibition of specific binding in ERα steroid ERβ steroid E4 finalbinding assay binding assay concentration Test 1 Test 2 Test 3 Test 1Test 2 Test 3   1 μM 98 nd nd 87 90 95 0.3 μM 92 94 101  74 74 77 0.1 μM83 85 86 56 54 50 0.03 μM  64 66 63 19 25 30  10 nM 43 32 28 nd nd nd  3 nM 26 17 11 nd nd nd nd: not determined

TABLE 2 Experimentally determined inhibition constants (Ki) for estetrol(E4), 17α-ethinylestradiol (EE) and 17β-estradiol (E2), to human ERα andERβ proteins. Steroid ligands Ki ERα (nM) Ki ERβ (nM) EE 0.23 0.025 E20.21 0.015 E4 4.9 19

Example 2

To determine the in vivo stability of estetrol (E4) its eliminationhalf-life in female Sprague Dawley rats was determined after a singlesubcutaneous administration (sc) at several dose levels.

Female Sprague Dawley rats were equipped with a permanent silatic heartcatheter, as described by Kuipers et al. (1985, Gastroenterology, 88,403-411). Rats were allowed to recover from surgery for 5 days and werethan administered 0.05, 0.5, or 5 mg/kg E4 in 0.5 ml arachidis oil. E4was injected in the neck area using a 1 ml syringe and 20 g needle.Blood samples were subsequently collected via the heart catheter inheparinized tubes at 0.5, 1, 2, 4, 8 and 24 hours. Erythrocytes wereremoved by centrifugation at 5000×g for 10 minutes at 4° C. and bloodplasma was stored at −20° C. After thawing the plasma samples,liquid-liquid extraction (hexane and diethyl ether) was employed toprepare the E4-containing plasma samples for HPLC analysis (Perkin Elmer200) and tandem mass spectrometry using a PE Sciex 3000 tandem massspectrometer and APCI interface. With each sample batch, a calibrationcurve with 6 calibrators was recorded. The calibration curve wascalculated using linear regression (correlation coefficient>0.98), whichpermitted quantitation of plasma concentrations. For each rat plasma,sampled at different time intervals, data were collected.

Plasma E4 concentration data were analysed with “WinNonLin, edition 3.1”and involved pharmacokinetic parameters for C_(max), AUC₀₋₂₄ andhalf-life. Interestingly, E4 demonstrated good stability with arelatively long half-life of 2-3 hours, enabling the detection ofbioactive levels of unconjugated E4 at all time points over a 24 hourinterval.

Example 3 Preparation of a Skin Cream Containing 1 mg/g Estetrol

A. Preparation of the Cream:

-   -   I. Boil in a beaker 60 ml purified water. Stop heating and add 4        grams solutio sorbitoli and 0.2 gram acidum sorbicum and        dissolve completely. Cover the beaker and cool down until 70° C.    -   II. Simultaneously heat 15 grams Cera cetomacrogolis        emulsificans together with 20 grams of Cetiol V until 70° C.    -   Mix I and II by stirring gently and allow the cream to cool down        (under occasional stirring). Add purified (boiled) water until        100 gram under stirring.        B. Preparation of the Cream Containing Estetrol:    -   Take 99.9 grams of the cream, as described under A, and add 100        mg of estetrol under stirring. Continue stirring until the        estetrol has been homogeneously dispersed throughout the cream.

Example 4

Twenty women, who are post-menopausal and in good health, are selectedon the basis of presenting several signs of rapid dermal atrophy, suchas a rapid increase in the number of facial wrinkles or crow's feet,rapid change in the pigmentation of the skin, i.e. “age spots”, or othercomplaints of rapid dermal ageing. Since dermal atrophy may also be theresult of other factors such as UV damage from the sun or otherenvironmental insults, consideration is taken to exclude patients, whoare suffering from these effects, from the clinical study.

The first component of the study is qualitative by making an evaluationof improvement in the patient's appearance. This evaluation requires abaseline level for future comparison. An initial baseline value iscreated in the form of a standardised set of questions as to how thepatient views her own appearance, photographs of the patient, and apsychological profile of the patient's self-image. The second componentis quantitative, including the measurement of urinary excretion ofhydroxyproline, moisture content of the skin, glycosaminoglycans in theskin, and changes in resilience and pliability of the skin. Methods fordetermining these factors are found in “The Menopause”, Ed. R J. Beard,University Press, Chapter 7 (1977) and “Methods in Skin Research”, Ed.Skerrow, D. and Skerrow C. J., John Wiley & Sons Ltd., Chp. 22,“Analysis of Sebaceous Lipids”, p. 587-608 (1985), and furtherreferences cited therein, all herein incorporated by reference. Again,before the start of the study baseline values of these quantitativefactors are obtained.

The study volunteers are subsequently placed in a clinical protocolreceiving the topical compound formulation as set forth in example 3.The compound formulation is administered to areas of the skin mosteffected by atrophy twice a day, which is continued for 1 to 3 months.Evaluations, both quantitative and qualitative, are made at biweeklyintervals.

The compound formulation, as set forth in example 3 gives positiveresults in a majority of participating study subjects by improving theoverall qualitative index of the patient's appearance and/or thequantitative parameters, e.g., an increase in the urinary excretion ofhydroxyproline signifying an increase in turnover and synthesis ofcollagen, an increase in moisture content, glycosaminoglycans,pliability, or resilience of the skin.

Example 5

Twenty women suffering from vaginal atrophy and/or vaginal drynesssymptom associated with menopause are selected. These women are ingeneral good health. Since the nature of this disorder is highlysubjective, evaluation of the effectiveness of treatment is necessarilyalso subjective in nature. The female subjects are asked to keep a dailylog, noting details as to the degree of vaginal itching, dryness anddyspareunia, and to use a visual analogue scale to record theirsubjective estimates. The change from pretreatment (base line value)during 1 to 3 months of treatment is assessed and considered to beindicative of the treatment efficacy.

The study volunteers subsequently participate in a clinical protocolreceiving the topical compound formulation as set forth in example 3.The compound formulation is administered intravaginally once a day,which is continued for 1 to 3 months. Evaluations of vaginal itching,dryness and dyspareunia are made at biweekly intervals. Utility ofestetrol is demonstrated by the positive results observed in a majorityof participating study subjects showing improvements in qualitativechange from baseline value for vaginal itching, dryness and dyspareunia.

Example 6 Wound Care Ointment Containing 1 mg/g Estetrol

Heat together 2.5 grams alcohol cetostearliylicus, 6 gram adeps lanae,51.5 gram vaselinum album and 40 grams paraffinum liquidum until allingredients are melted. The warm mixture is filtered using a paperfilter. The filtered mixture is subsequently sterilised using a 0.2micrometer filter.

100 mg estetrol is thoroughly mixed with 99.9 gram of the abovedescribed ointment under aseptic conditions.

The invention claimed is:
 1. A cosmetic method of improving wrinkled,lined, dry, flaky, aged, or photodamaged skin comprising applying to theskin a composition comprising: (i) at least 5 μg of estetrol per g ofcomposition; and (ii) a cosmetically acceptable vehicle.
 2. The cosmeticmethod according to claim 1, wherein the composition enhancesproliferation of fibroblasts and/or epidermal skin cells.
 3. Thecosmetic method according to claim 1, wherein the composition improvesskin thickness and/or skin elasticity.
 4. The method according to claim1, wherein the composition contains at least 10 μg/g of estetrol.
 5. Thecosmetic method according to claim 1, wherein the composition is appliedto facial skin or neck skin.
 6. The cosmetic method according to claim1, wherein the composition is applied at least once a day during aperiod of at least 3 days.
 7. The cosmetic method according to claim 1,wherein virtually all of the composition that has been applied to theskin penetrates the skin.
 8. A method of promoting wound healingcomprising applying to a wounded tissue or to tissue in a vicinity of awound a composition comprising: (i) at least 5 μg of estetrol per g ofcomposition; and (ii) a cosmetically acceptable vehicle.
 9. A method oftreating or preventing acne comprising applying to skin that is affectedby acne or that is at risk of being affected by acne a compositioncomprising: at least 5 μg of estetrol per g of composition; and (ii) acosmetically acceptable vehicle.
 10. A pharmaceutical or cosmeticcomposition for topical administration comprising: at least 5 μg ofestetrol per g of composition; and (ii) a cosmetically acceptablevehicle.
 11. The cosmetic method according to claim 1, wherein thecomposition is a lotion, salve or a cream.